Abstract Abuse of the highly addictive psychostimulant methamphetamine (meth) is a large and growing public health concern. Meth abuse has devastating social and economic consequences, costing the U.S. billions each year. At present, there are no effective pharmacotherapies for the treatment of meth addiction. The objective of this proposal directly addresses this unmet clinical need, as it includes the evaluation of a novel anti-abuse therapeutic strategy. Meth, like other reinforcing substances, results in aberrant activation of the mesolimbic dopamine system. Restoring dopamine normality in this system is, thus, indicated as a potential addiction treatment strategy. Restoration of dopamine signaling homeostasis may be achieved by targeting the G-protein- coupled receptor (GPCR) neurotensin receptor 1 (NTR1). The efficacy of peptide NTR1 agonists in animal models of addiction have made drug-like, small molecule NTR1 ligands highly sought-after. NTR1, like other GPCRS, signals through both G-protein- and ?-arrestin-mediated pathways. Because of the historical lack of success identifying NTR1 agonists in G protein-based assays, a large-scale screen and optimization effort was undertaken to identify small molecule NTR1 ligands in a ?-arrestin screen. We have characterized the current lead compound SBI-553 as a ?-arrestin biased NTR1 ligand, as it activates ?-arrestin without stimulating NTR1 G-protein signaling. Promising preliminary data show that SBI-553 attenuates meth-associated behaviors in mice. The objectives of this application are to elucidate the mechanism of action of ?-arrestin biased NTR1 ligands and validate their therapeutic potential in clinically relevant murine models of meth use. The central hypothesis is that ?-arrestin biased agonism at NTR1 stimulates NTR1 trafficking and signaling to attenuate the physiological and behavioral consequences of meth exposure. This hypothesis will be tested by pursuing three specific aims, using SBI-553 as a tool compound: 1) Identify the biochemical mechanism of action of ?-arrestin biased NTR1 ligands, 2) Elucidate the physiological effects of ?-arrestin biased NTR1 ligands, and 3) Elucidate the behavioral effects of ?-arrestin biased NTR1 ligands. These aims will be addressed using established GPCR trafficking and signaling assays, state-of-the-art small animal positron emission tomography (PET)/computed tomography (CT) imaging, and murine behavioral studies, including self-administration, in wild-type animals and those lacking NTR1 (NTR1-/-). Exploiting GPCR signaling bias in the treatment of disease is in its infancy, making this application both timely and novel. These studies are significant in that they will not only advance our understanding of how the NT system regulates addictive behaviors, but may also facilitate the development of novel anti-abuse agents and improve outcomes for patients struggling with chemical addiction.